Optical arrangement and method for imaging a sample

ABSTRACT

An optical arrangement for imaging a sample is disclosed. The optical arrangement comprises at least one first objective lens and at least one second objective lens, at least one illumination source for producing an illumination beam, detector for imaging radiation from the sample, and at least one mirror for reflecting the radiation from one of the first objective lens or the second objective lens into the detector. The at least one mirror is double-sided and dependent on the illumination beam at the other one of the first objective lens and the second objective lens.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation under 35 USC § 120 of U.S. patent applicationSer. No. 15/302,569 filed Oct. 7, 2016, which in turn is a U.S. nationalphase application under 35 U.S.C. § 371 of International PatentApplication No. PCT/EP15/57576 filed Apr. 8, 2015, which in turn claimspriority of European Patent Application No. 14163919.5 filed Apr. 8,2014. The disclosures of U.S. patent application Ser. No. 15/302,569,International Patent Application No. PCT/EP15/57576, and European PatentApplication No. 14163919.5 are hereby incorporated herein by referencein their respective entireties, for all purposes.

FIELD OF THE INVENTION

The invention relates to an optical arrangement and a method for imaginga sample using at least two illumination beams.

BACKGROUND TO THE INVENTION

A microscope is a scientific instrument that is used for thevisualization of objects, which can be either small cells or havedetails that are too small to be resolved by the naked eye.

There are many types of microscopes available on the market. The mostcommon of these and the first to be invented is the so-called opticalmicroscope, which uses light in a system of lenses to magnify images ofthe samples. The image from the optical microscope can be either viewedthrough an eyepiece or, more commonly nowadays, captured by alight-sensitive camera sensor to generate a so-called micrograph. Thereare a wide range of sensors available to catch the images. Non-limitingexamples are charge-coupled devices (CCD) and scientific complementarymetal-oxide semiconductor (sCMOS) based technologies, which are widelyused. These sensors allow the capture and storage of digital images tothe computer. Typically there is a subsequent processing of these imagesin the computer to obtain the desired information.

The illumination sources as used in optical microscopes have beendeveloped over the years and wide varieties of illumination sources arecurrently available, which can emit light or other type of radiation atdifferent wavelengths. Optical filters can be placed between theillumination source and the sample to be imaged in order to restrict thewavelength of the radiation illuminating the sample.

Modern biological microscopy uses fluorescent probes for imagingspecific structures within a cell as the sample. In contrast to normaltrans-illuminated light microscopy, the sample in fluorescent microscopyis illuminated through one or more objective lenses with a narrow set oflight wavelengths. These narrow set of light wavelengths interact withfluorophores in the sample, which then emit light of a differentwavelength. This emitted fluorescent light is detected in a detector andis used to construct the image of the sample.

The use of multiple images enables a 3-dimensional reconstruction of thesample to be made. This 3-D reconstruction can be done by generatingimages at different positions on the sample, as the sample movesrelatively to one or more objective lens. Depending on the number ofdetection units necessary, several detectors may be required. Thesedetectors are quite expensive and a microscope designer will wish toreduce the number of detectors. The use of a single detector, which ismoved during the imaging process, can be disadvantageous in that themovement of the detector itself can slightly effect the position of thesample, due to vibrations. Alternately the sample itself may move forother reasons whilst the detector is being placed into another position.This movement of the detector requires a precise and fast movement of apart of hardware, which is comparatively massive and in turn leads tofurther increase in development costs and/or in extra parts ofequipment.

A number of papers and patents have been published on various aspects ofmicroscopy. For example, European patent EP 1 019 769 (Carl Zeiss, Jena)teaches a compact confocal feature microscope, which can be used as amicroscope with a single objective lens or with multiple objectivelenses. The microscope has separate directions of illumination anddetection. The direction of detection in the objective lens is alignedinclined at a set angle in relation to the direction of illumination.

Another example of a microscope is taught in the paper by Krzic al.“Multi View Light-Sheet Microscope for Rapid in toto Imaging”, NatureMethods, July 2012, vol. 9 No. 7, pages 730-733. This paper teaches amulti-view selective-plane illumination microscope comprising twodetection and illumination objective lenses. The microscope allows intoto fluorescence imaging of the samples with subcellular solution. Thefixed geometrical arrangement of the imaging branches enables multi-viewdata fusion in real time.

Document DE 195 09 885 A1 discloses a stereo endoscope whereinilluminating light is transmitted by the light guide inserted throughthe elongate inserted section and is projected out of the distal endsurface of the inserted section. The illuminated objects pass throughthe respective pupils of the two objective lens systems arranged inparallel within the distal end section of the inserted section and theirimages are formed on the focal surface. The respective images aretransmitted to the rear side by one common relay lens system. Thetransmitted final images are formed respectively on the image takingsurfaces of the image taking devices. The respective images arephotoelectrically converted by the respective image taking devices andfurther processed to be signals, are displayed in the monitor and arestereo-inspected through shutter spectacles.

Document U.S. Pat. No. 4,440,475 A discloses a device having aelectromagnetic lens for focusing the analyzing electron beam that isprovided with a central channel along the axis of the electron beamwhich is intended to pass through a mirror-objective having highmagnification. The electromagnetic lens further comprises a lateralchannel in which it is placed an auxiliary objective having lowmagnification. An optical illumination system, the axis of which iscontained in the plane of the axes of the objectives, illuminates thesample either through the principal objective or through the auxiliaryobjective. An orientable mirror which is orthogonal to the planeaforesaid and placed at the intersection of the beams which form theimages through the two objectives permits the use of the sameobservation means both for low magnification and for high magnification.

In document U.S. Pat. No. 5,132,837 A is an operation microscopedisclosed including a plurality of objective lenses arranged atdifferent angles with respect to an object to be viewed and a selectingoptical system having a function of selecting one of light beams fromthe objective lenses and enabling the object to be observed at thedifferent angles. Accordingly, a visual field for observation of anobject to be operated may be expanded.

Document US 2012/044486 A1 discloses a system and a method for detectingdefects on a waver.

Document WO 2008/028045 A2 discloses a system and method for robustfingerprint acquisition comprising combined multispectral andtotal-internal-reflectance biometric imaging systems. A platen hasmultiple facets, at least one of which has a surface adapted forplacement of a purported skin site by an individual and another facetmay include an optical absorber. An illumination source and an opticalarrangement are disposed to illuminate the purported skin site withlight from the illumination source along distinct illumination paths,including paths at angles less than the critical angle and paths atangles greater than the critical angle. Both multispectral andtotal-internal-reflectance illumination are received by an imagingsystem. The imaging system may include first and second imaginglocations adapted to record images from separate illumination paths. Theplaten may also include non parallel exits facets

SUMMARY OF THE INVENTION

An optical arrangement for imaging a sample is disclosed. The opticalarrangement comprises at least one first objective lens and at least onesecond objective lens, at least one illumination source for producing anillumination beam, a detector for imaging radiation from the sample, andat least one mirror. The at least one mirror is adapted to reflectingthe radiation from one of the first objective lens or the secondobjective lens into the detector. The position of the mirror isdependent on the illumination beam at the other one of the firstobjective lens and the second objective lens wherein the mirror can bedouble-sided. The use of the double-sided mirror enables a singledetector to be used to image the sample from multiple sides and thussave on the detectors.

In one aspect of the disclosure the at least one mirror is translatableor rotatable.

In another aspect of the disclosure at least two mirrors are present andthe reflected radiation can be directed into one of the at least twomirrors.

An optical filter can be inserted in the path of the illumination beamor the path of the radiation to select only certain wavelengths oflight.

In a further aspect of the invention, a third objective lens can be usedto collect the radiation from the sample.

A method for imaging a sample is also disclosed. The method comprisesilluminating the sample using a first illumination beam, detecting, at astationary detector in a stationary position, first radiation from thesample, processing the first radiation to obtain a first data set,illuminating the sample using a second illumination beam at an angle tothe first illumination beam, detecting, at the stationary detector inthe stationary position, second radiation from the sample, processingthe second radiation to obtain a second data set, and combining thefirst data set and the second data set to produce an image of thesample.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic view of an optical arrangement according tothis disclosure.

FIG. 2a shows a first aspect of the optical arrangement.

FIG. 2b shows another position of the first aspect of the opticalarrangement.

FIG. 3a shows a second aspect of the optical arrangement.

FIG. 3b shows another position of the second aspect of the opticalarrangement.

FIG. 4 shows a method imaging a sample according to this disclosure.

FIG. 5a shows a third aspect using more than two objective lenses.

FIG. 5b shows another position of the third aspect of the opticalarrangement

FIG. 5c shows another position of the third aspect of the opticalarrangement

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described on the basis of the drawings.

It will be understood that the embodiments and aspects of the inventiondescribed herein are only examples and do not limit the protective scopeof the claims in any way. The invention is defined by the claims andtheir equivalents. It will be understood that features of one aspect orembodiment of the invention can be combined with the feature of adifferent aspect or aspects and/or embodiments of the invention.

FIG. 1 shows an overview of an optical arrangement 10 of thisdisclosure. The optical arrangement 10 has a first objective lens 30 anda second objective lens 40. Both the first objective lens 30 and thesecond objective lens 40 are able to image a sample 20 and/or direct anillumination beam 60 and 60′ onto the sample 20. The optical arrangement10 shown in FIG. 1 has two objective lenses 30 and 40, but this is notlimiting of the invention. It would be possible to have an opticalarrangement with a larger number of objective lenses.

The optical arrangement 10 has an illumination source 50 that producesthe illumination beam 60. The optical arrangement 10 has also a detector70 that is able to detect radiation 80 reflected or fluoresced from thesample 20.

The sample 20 is typically a biological sample. The sample 20 is to beimaged in three dimensions. It is known that a minimum of one view isrequired to create a 3D stack of images. At least two views are requiredin order to make a multi-view image of the sample 20 in the opticalarrangement of FIG. 1a . The views can then be stored in a memory 110 asa first data set 120 and a second data set 130 and combined in aprocessor 100 in order to construct a multidimensional dataset. Thismultidimensional dataset can, for example, be used to create a 3Dmulti-view image of the sample 20.

Both of the first objective lens 30 and the second objective lens 40 canbe used to illuminate the sample 20 and/or gather radiation fluorescedor reflected from the sample 20. This will be described using a blackbox 95 as is illustrated in FIG. 1. The black box 95 outlines the mannerin which the illumination beam 60 from the illumination beam source 50can be directed either to the first objective lens 30 or, for example byuse of mirrors, as an illumination beam 60′ to the second objective lens40. The black box 95 also shows that radiation from the sample 20 can bedirected either through the first objective lens 30 as a radiation beam80′ or from the second objective lens 40 and thence to the detector 70as a radiation beam 80. The optical arrangement 10 illustrated in FIG. 1is therefore able to create at least two images of the sample 20 fromdifferent angles in order to allow the construction of a 3D image of thesample 20. This principle can be implemented using different mirrorarrangements as described below.

FIGS. 2a and 2b show a first aspect of the optical arrangement 10 inwhich the illumination source 50 is located to one side of the detector70. In FIG. 2a , the illumination source 50 is shown to the left handside of the single detector 70 and produces an illumination beam 60arriving at the first objective lens 30 from which the illumination beam60 is projected onto the sample 20. Radiation from the sample 20 isimaged through the second objective lens 40 and strikes the right mirror90 a on the right hand side from which the radiation 80 is reflected toa central mirror 90 c and thence into the detector 70. FIG. 2a includesfurther a left mirror 90 b. It will be seen from FIG. 2a that the lefthand mirror 90 b does not interrupt the passage of the illumination beam60 from the illumination source 50 on the left hand side.

The optical arrangement 10 of FIG. 2a can be compared with the opticalarrangement 10 shown in FIG. 2b . The optical arrangement 10 of FIG. 2bcomprises the same elements with the same numbers as shown in FIG. 2a .FIG. 2b shows, however, a further illumination source 50′ on the righthand side which produces an illumination beam 60′ entering the secondobjective lens 40. It will be seen that the right hand mirror 90 a hasbeen moved out of the path of the illumination beam 60′, so that thisright hand mirror 90 a does not interrupt the passage of theillumination beam 60′. The radiation 80′ from the sample 20 passesthrough the first objective lens 30 and is reflected by the left-handside mirror 90 b onto the central mirror 90 c and thence into thedetector 70. It will be noted that the right hand mirror 90 a, the lefthand mirror 90 b and the central mirror 90 c have in FIG. 2b beenshifted to the right compared to the equivalent positions in FIG. 2a inorder to allow the illumination beams 60, 60′ and the radiation 80 and80′ to be reflected differently. This is indicated figuratively by arrow92. It will be understood that the movement of the mirrors 90 a, 90 b,90 c, as indicated by the arrow 92, and can be easily implemented, forexample on a sliding track.

FIGS. 3a and 3b show a second aspect of the invention in which a singlecentral mirror 90 c is moved up and down, as indicated by an arrow 94.The optical arrangement 10 of FIGS. 3a and 3b has otherwise the sameelements as the optical arrangement shown on FIGS. 2a and 2b . Theoptical arrangement 10 has, however, a single detector 70 on the lefthand side and a single illumination source 50 on the right hand side.

FIG. 3a shows a lower position of the central mirror 90 c in which theillumination source 50 produces the illumination beam 60′ reflected bythe central mirror 90 c onto the right hand mirror 90 a and thence intothe second objective lens 40, thereby illuminating the sample 20.Radiation from the sample 20 is collected by the first objective lens 30and reflected by the left hand mirror 90 b onto the central mirror 90 cand thence into the detector 70.

In the aspect shown in FIG. 3b the central mirror 90 c is moved to makeway for the illumination beam 60 and the radiation 80. In this examplethe illumination beam 60 is produced by the illumination source 50 andis reflected by the left hand mirror 90 b onto the sample 20 through thefirst objective lens 30. The radiation from the sample 20 is imagedthrough the second objective lens 40 and is reflected by the right handmirror 90 a into the detector 70. Using the aspect of the inventionshown in FIGS. 3a and 3b two images of the sample 20 can be produced.

The method of the invention is shown in FIG. 4 in which an illuminationbeam is produced in step 200 from the illumination source 50 or 50′. Itwill be understood from FIGS. 2a and 2b as well as FIGS. 3a and 3b andFIGS. 5a-c that there may be a single one of the illumination source 50(as shown in FIGS. 3a and 3b and FIFA. 5 a-c) or two illuminationsources 50 and 50′ as shown in FIGS. 2a and 2b . There may be moreillumination sources, as is the case of having more than two objectivesas depicted in FIGS. 5 a-c. The illumination beam 50 is directed in step205 to the sample 20 and reflected in step 206 from the sample 20. Thedetection of the radiation 80 is carried out in step 210 in the detector70. The detector 70 can be, for example, a charge coupled device or anyother detection instrument.

A first image produced from the illumination beam is processed in step220 in a processor 100 and stored in a memory 110 as a first data set120.

A second illumination beam coming from an illumination source 50 iscreated in step 230 and illuminates the sample 20 from a differentdirection in step 235. The second illumination beam is reflected fromthe sample 220 in step 238 and is detected in the same detector 70. Theimage is then processed in the processor in step 250 and stored in thememory 110 as a second data set 130. The first data set 120 and thesecond data set 130 forming the two images can be combined in step 260in the processor 100 to produce the multi-view 3D image of the sample20.

FIGS. 5a-c shows a third aspect of this disclosure using more than twoobjective lenses. The illumination beam 60, 60′ or 60″ is produced fromthe illumination source 50 and passes through one of a plurality ofoptical selectors 91 a, 91 b or 91 c. The optical selectors 91 a, 91 bor 91 c may be optical filters allowing passage of certain wavelengths,movable mirrors, moving shutters, or another possible optical selectordevice. FIGS. 5a-c illustrates particular cases in which an opticalfilter that allows certain wavelengths to pass through is utilized forthe optical selectors 91 a, 91 b, or 91 c. The illumination beam 60″ ofthe aspect depicted in FIG. 5a passes through the optical filter 91 c.The illumination beam 60″ passes through a third objective lens 31 andilluminates the sample 20. The radiation 80 coming from sample 20 iscollected by the second objective lens 40, reflects on the opticalselector 91 b, and reflects on the mirror 90 b towards a radiationselector 96. The radiation selector 96 is shown as a rotating mirror inFIGS. 5a-c . The radiation selector 96 can be another type of movableradiation redirecting devices. The redirected radiation 80, 80′ or 80″is directed onto the detector 70.

In this aspect of the invention each one of the first, third or secondobjective lenses 30, 31 and 40 can be used for illumination ordetection. It is therefore also possible to collect the radiation 80′from sample 20 with the first objective lens 30, reflecting theradiation 80′ on the optical selector 91 a and on the mirror 90 a, andsubsequently causing the radiation selector 96 to redirect the radiation80′ to the detector 70, as demonstrated in FIG. 5 c.

It is also possible to illuminate the sample 20 by sending anillumination beam 60 and/or 60′ either through the optical selector 91 aand/or the optical selector 91 b, through the first objective lens 30and/or the second objective lens 40, and collecting the radiation 80″from the third objective lens 31, reflecting the collected radiation 80″with the optical selector 91 c and further with mirror 90 c onto theradiation selector 96, thus redirecting radiation 80″ to detector 70, asdemonstrated in FIG. 5 b.

REFERENCE NUMERALS

-   10 Optical arrangement-   20 Sample-   30 First objective lens-   31 Third objective lens-   40 Second objective lens-   50, 50′ Illumination source-   60, 60′, 60″ Illumination beam-   70 Detector-   80, 80′, 80″ Radiation-   90 a,b,c Mirror-   91 a,b,c Optical Selector-   92 Arrow-   94 Arrow-   95 Black box-   96 Radiation selector-   100 Processor-   110 Memory-   120 First data set-   130 Second data set

The invention claimed is:
 1. An optical arrangement for imaging asample, comprising: an illumination source; a detector; a movableradiation selector; a plurality of light paths, ones of the plurality oflight paths comprising an objective lens; an illumination light path forpassing an illumination beam from the illumination source through theobjective lens to illuminate the sample; a detection light path forpassing radiation collected from the sample through the objective lensto the detector to image the sample; wherein the detection light pathsmeet at the movable radiation selector, and the movable radiationselector is configured to direct, in a predefined position of themovable radiation detector, the detection light path of a selected oneof the plurality of light paths towards the detector.
 2. The opticalarrangement of claim 1, wherein the radiation selector is a rotatingmirror.
 3. The optical arrangement of claim 1, wherein one or more ofthe plurality of light paths comprise an optical selector, arranged inthe illumination light path and in the detection light path, forselectively allowing passage of the illumination beam towards the sampleand for reflecting the radiation collected from the sample towards thedetector.
 4. The optical arrangement of claim 1, wherein theillumination light path and the detection light path of the ones of theplurality of light paths partially overlap.
 5. The optical arrangementof claim 1, wherein at least one of the plurality of light paths ischosen to illuminate the sample, and at least another distinct one ofthe plurality of light paths is chosen to image the sample.
 6. Theoptical arrangement of claim 1, wherein the ones of the plurality oflight paths further comprises a mirror to reflect the detection lightpath towards the radiation selector.
 7. A method of imaging a sample bymeans of a plurality of light paths, the method comprising selecting atleast one illuminating one of the plurality of light paths forilluminating the sample selecting at least one detecting one of theplurality of light paths for detecting radiation from the sample,wherein the selecting of the at least one detecting one of the pluralityof light paths comprises positioning in a predefined position a movableradiation selector, passing, by means of the at least one illuminatingone of the plurality of light paths, an illumination beam from a lightsource via an illuminating objective lens to the sample, passing, bymeans of the at least one detecting one of the plurality of light paths,radiation from the sample via a detecting objective lens and via themovable radiation selector to the detector for detecting the radiation,processing the radiation to create first image data wherein the movableradiation selector is configured to direct the detecting one of theplurality of light paths towards the detector.
 8. The method of claim 7,further comprising repeating the steps of the method to create secondimage data, and combining the first image data with the second imagedata to produce an image of the sample.
 9. The method of claim 7,wherein the movable radiation selector is configured to reflectradiation towards the detector.